Effect of Food Predictability on Life Span in Male Mice.

The purpose of this study is to compare the effect of unpredictable (U) or predictable (P) food delivery on health and longevity in mice. From 2 months of age until end of life, singly-housed male C57BL/6 mice were fed a semisynthetic diet either ad libitum (AL), or as imposed meals delivered as small pellets at either P or U times, frequencies, or amounts. The total daily food consumed by all groups was the same. The AL group gained body weight faster than either P or U groups, and had ~12% shorter median life span compared with either P or U groups. Bimonthly noninvasive body composition determinations showed that the differences in body weights were due to differences in fat and lean mass. Postmortem examinations revealed that the organ pathologies were similar in all groups, but a larger fraction of P and U mice were euthanized due to end-of-life suffering. There were no systematic differences in outcome measures between P and U groups suggesting that, within the range studied, the temporal pattern of food delivery did not have a significant metabolic effect.

Temporal relationships between food acquisition and voluntary exercise in mice.

Patterns of operant food acquisition in a closed economy and bouts of either voluntary wheel running (WR) or spontaneous locomotor activity in a standard condition (SC) with no wheel were examined in young adult male and female C57BL/6 mice across a range of nose poke prices (FUP) per food pellet. Both sexes showed vigorous WR or locomotor activity. At each FUP, WR groups had higher food intake than SC groups. Despite substantially higher mean body weight of males compared with females, intakes and activity did not differ by sex in the SC groups and males lost weight more rapidly as FUP increased. In contrast, WR males ran ∼33% further per day than females, increased their food intake (above that of SC counterparts) more than females, and lost less body weight than SC males. By parsing the night in four 3h epochs it was found that food intake declined progressively through the night in both WR and SC mice and that the hyperphagia of WR relative to SC groups was most evident early in the night, coincident with highest activity. No large or systematic sex differences were revealed in these temporal analyses. Analysis of data at 60s resolution showed that pellet acquisition occurred in many small or short bouts, the timing of which was either intercalated or concurrent with either locomotor activity or WR. The results show that increased eating due to WR occurs concurrently with maximum running, and with no evidence of delayed compensation.

Circadian and economic factors affect food acquisition in rats restricted to discrete feeding opportunities.

The purpose of this study is to examine aspects of operant behavior-modeled economic choice for food in rats in closed economy protocols in which food is available for only a few discrete times per daily 23-h session, designed to emulate clustering of human food intake into meals. In the first experiment, rats performed lever press responses for food pellets in an ascending series of ratios or fixed unit prices (FUP) when food was available for four 40-min food opportunities (FO) per day. Daily intake at low FUP was comparable to ad libitum intakes. Intake declined as FUP increased and was not distributed equally among the four FOs. In particular, the last FO of a session (occurring at about lights on in a 12:12cycle) was the smallest, even when total intake was low due to the response requirement at high FUP. Within FOs, satiation was evident at low FUPs by a decrease in rate of intake across a 40min FO; at high FUPs responding was evenly distributed. In the second experiment, rats had a choice of responding on two levers for either intermittent inexpensive (II; low FUP according to a four FO schedule) or costly continuous (CC; 20-fold higher FUP but available throughout 23-h sessions) food. Most (73%) of the rats consistently chose almost all of their food from the II source. Further, as the timing of the four II FOs were changed relative to the light: dark Zeitgeber, the time of the smallest meal changed such that the smallest meal (s) were during the light period regardless of ordinal position within a session. These data are discussed in terms of economic and Zeitgeber effects on consumption when food is available intermittently, and are contrasted with results from comparable protocols in mice.

Effect of day-night cycle on distribution of food intake and economic choice among imposed food opportunities in mice.

We have shown previously that mice given access to four discrete feeding opportunities (FOs) per day show a characteristic sequence of sizes across ordinal FOs. The purpose of the present experiments was to determine the relative contributions of external and internal factors on the sequencing of FO size. The external factors were the light:dark Zeitgeber and the cost of food, imposed via different fixed unit prices (FUP) in a closed operant economy, and the internal factors were signals relating to energy status including time since last food and weight loss. In the first experiment, mice were given 4 FOs spaced 4-h apart, but with the timing of the FOs relative to the Zeitgeber altered by a 4-h Zeitgeber advance or delay of the cycle. Food intake, and associated body weight, declined as price increased, but the temporal order of FO size was invariant within a Zeitgeber condition. The Zeitgeber advanced group showed clear evidence of a shift in meal sequence relating to the light:dark cycle. Thus, external factors seem to be a more important determinant of total intake and sequencing than internal factors. In the second experiment, mice were given the choice between continuous costly (CC) and intermittent inexpensive (II) food. II food was available for four-15min intervals every 4-h, and the timing of the 15min intervals was varied relative to the Zeitgeber cycle. In spite of a 20-fold difference in price between CC and II food, mice took approximately equal amounts from each, and all food intake took place during the dark phase. Mice consumed II food only if it was available during the dark phase. Food intake was strongly linked to the light:dark cycle, largely independent of food cost.

Restricted temporal access to food and anorexia in mice: Microstructure of eating within feeding opportunities.

Intake and body weight were recorded in a closed economy as male and female C57BL/6 mice progressed through either fixed interval (FI) or fixed unit price (FUP) schedules of cost for 20-mg food pellets. Access to food was constrained to four 40 min food opportunities (FOs) per day, spaced 4-h apart through the dark phase. Nose poke responses and pellet deliveries were collected at 10-s resolution to allow pellet-by-pellet analysis. In the FI protocol, mice maintained adequate food intake and body weight through the study, even though at the highest FI (50-s) they spent the entire 40-min FOs engaged in eating at or near the maximum rate allowed by the schedule. In the FUP protocol, mice greatly reduced their intake and lost weight at the highest FUP (50 responses/pellet). The analysis of response and pellet distributions showed these mice were not filling the FOs with responding and ate less at dusk (FO #1) and dawn (FO #4) than at FOs #2 and 3 in the middle of the night. The principal, and unexpected, sex difference was that females tended to eat more than males despite lower body weight, but behavioral changes as a function of feeding cost or schedule were qualitatively similar in both sexes. These results show that slow eating as imposed by an FI is not sufficient to produce hypophagia and, in the FUP protocol, hypophagia cannot be explained by slowed eating due to response requirements. We discuss the role of effort or time in FUP-induced anorexia, and suggest this murine model may emulate some aspects of human anorexia nervosa better than current activity-based protocols.

Differences in temporal aspects of food acquisition between rats and two strains of mice in a closed operant economy.

Rats and mice were studied for changes in meal-taking structure in a closed operant food economy, in which the consummatory or unit prices for food were increased. In experiment 1, as food price increased, male rats modestly decreased the number of meals per day and increased mean meal size. Female rats were similar to males but had smaller meal size and, at low costs, took more meals per day. In experiment 2, male and female B6 mice reduced food intake as price increased, accompanied by decreased meal number without change in meal size. They showed grazing-like behavior in the first part of the night. In contrast, we report in experiment 3, a large increase in intake and meal size during the final trimester of pregnancy. In experiment 4, we report that CD1 male mice subjected to a unit price series performed comparably to rats, and not like B6 mice. Other CD1 mice were tested using an interval schedule, and we found that mice were able to adapt eating patterns to greatly increased time demands without compromising total intake. Data are discussed in terms of the intercalation of food acquisition with global patterns of activity. Such interactions of organism and food environment are in particular need of mechanistic investigation.

Role of estrogen receptor-α on food demand elasticity.

Estrogens have been shown to have an inhibitory effect on food intake under free-feeding conditions, yet the effects of estrogens on food-maintained operant responding have been studied to a much lesser extent and, thus, are not well understood. Therefore, the purpose of the present experiment was to use a behavioral economics paradigm to assess differences in demand elasticity between mice with knockout of the estrogen receptor subtype α, knockout of subtype ß, and their wild type controls. The mice responded in a closed economy, and the price of food was increased by increasing the fixed-ratio response requirement every four sessions. Overall, we found that mice with the knockout of receptor subtype α had the most elastic demand functions. Therefore, under these conditions, estrogens increased food seeking via activation of the receptor subtype α. The results were inconsistent with those reported by previous studies that employed free-feeding conditions.

Effects of caloric restriction on nitrogen and carbon stable isotope ratios in adult rat bone.

RATIONALE: Stable isotope analysis is a valuable technique for dietary estimation in ecological and archaeological research, yet many variables can potentially affect tissue stable isotope signatures. Controlled feeding studies across a range of species have consistently demonstrated impacts of caloric restriction on tissue stable isotope ratios, but most have focused on juvenile, fasting, and/or starving individuals, and most have utilized soft tissues despite the importance of bone for paleodietary analyses. The goal of this study was to determine whether temporally defined, moderate food restriction could affect stable carbon and/or nitrogen isotope ratios in adult mammalian bone - a tissue that arguably reflects long-term dietary signals. METHODS: Adult rats fed a standard laboratory diet were restricted to 45% of ad libitum intakes for 3 or 6 months. Relevant anatomical and physiological parameters were measured to confirm that the restriction protocol resulted in significant nutritional stress and to provide independent data to facilitate interpretation of stable isotope ratios. Femoral bone δ(13)Ccollagen, δ(15)Ncollagen, and δ(13)Capatite values were determined by isotope ratio mass spectrometry. RESULTS: Calorie-restricted animals exhibited a small, yet significant enrichment in (15)Ncollagen compared with control animals, reflecting protein-calorie stress. While the δ(13)Ccollagen values did not differ, the δ(13)Capatite values revealed less enrichment in (13)C than in controls, reflecting catabolism of body fat. Independent anatomical and physiological data from these same individuals support these interpretations. CONCLUSIONS: Results indicate that moderate caloric restriction does not appreciably undermine broad interpretations of dietary signals in adult mammalian bone. Significant variability among individuals or groups, however, is best explained by marked differences in energy intake over variable timescales. An inverse relationship between the δ(13)Capatite and δ(15)Ncollagen values observed in this study indicates that a more robust pattern is expected with more severe or prolonged restriction and suggests this pattern may have utility as a marker of food deprivation in archaeological populations.

Economics of food intake in mice: energy yield of the reinforcer.

One of the Zeitgeists of the field for the study of ingestive behavior is that organisms are endowed with internal self-regulatory mechanisms that ensure optimal nutrition. However, the alarming increase in the prevalence of obesity challenges us to reconsider the extent to which internal regulatory mechanisms affect food intake, especially in a free market economy. Cued by the pioneering work of George Collier and his students, we have been examining food intake (demand) in mice when the effort or price of food is manipulated. We present two new experiments in mice that investigate the effect of energy yield per unit of food earned on working for food. The first experiment shows that when the nominal energy yield of each food pellet is halved by cellulose dilution, mice show relatively inelastic calorie-related demand despite the fact the cellulose diluted diet is unpalatable. The second experiment shows that the size of the pellet reinforcer does not have a major effect on food demand except in the extreme condition of small reward and high unit price. New analyses of distributions of responding are presented which suggest that mice work for "target" numbers of food rewards with only a small influence of price or energy gain.

A novel aminotetralin-type serotonin (5-HT) 2C receptor-specific agonist and 5-HT2A competitive antagonist/5-HT2B inverse agonist with preclinical efficacy for psychoses.

Development of 5-HT2C agonists for treatment of neuropsychiatric disorders, including psychoses, substance abuse, and obesity, has been fraught with difficulties, because the vast majority of reported 5-HT2C selective agonists also activate 5-HT2A and/or 5-HT2B receptors, potentially causing hallucinations and/or cardiac valvulopathy. Herein is described a novel, potent, and efficacious human 5-HT2C receptor agonist, (-)-trans-(2S,4R)-4-(3'[meta]-bromophenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine (-)-MBP), that is a competitive antagonist and inverse agonist at human 5-HT2A and 5-HT2B receptors, respectively. (-)-MBP has efficacy comparable to the prototypical second-generation antipsychotic drug clozapine in three C57Bl/6 mouse models of drug-induced psychoses: the head-twitch response elicited by [2,5]-dimethoxy-4-iodoamphetamine; hyperlocomotion induced by MK-801 [(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (dizocilpine maleate)]; and hyperlocomotion induced by amphetamine. (-)-MBP, however, does not alter locomotion when administered alone, distinguishing it from clozapine, which suppresses locomotion. Finally, consumption of highly palatable food by mice was not increased by (-)-MBP at a dose that produced at least 50% maximal efficacy in the psychoses models. Compared with (-)-MBP, the enantiomer (+)-MBP was much less active across in vitro affinity and functional assays using mouse and human receptors and also translated in vivo with comparably lower potency and efficacy. Results indicate a 5-HT2C receptor-specific agonist, such as (-)-MBP, may be pharmacotherapeutic for psychoses, without liability for obesity, hallucinations, heart disease, sedation, or motoric disorders.

Dehydration parameters and standards for laboratory mice.

Water deprivation and restriction are common features of many physiologic and behavioral studies; however, there are no data-driven humane standards regarding mice on water deprivation or restriction studies to guide IACUC, investigators, and veterinarians. Here we acutely deprived outbred CD1 mice of water for as long as 48 h or restricted them to a 75% or 50% water ration; physical and physiologic indicators of dehydration were measured. With acute water deprivation, the appearance and attitude of mice deteriorated after 24 h, and weight loss exceeded 15%. Plasma osmolality was increased, and plasma volume decreased with each time interval. Plasma corticosterone concentration increased with duration of deprivation. There were no differences in any dehydration measures between mice housed in conventional static cages or ventilated racks. Chronic water restriction induced no significant changes compared with ad libitum availability. We conclude that acute water deprivation of as long as 24 h produces robust physiologic changes; however, deprivation in excess of 24 h is not recommended in light of apparent animal distress. Although clearly thirsty, mice adapt to chronic water restriction of as much as 50% of the ad libitum daily ration that is imposed over an interval of as long as 8 d.

Molecular and behavioral pharmacology of two novel orally-active 5HT2 modulators: potential utility as antipsychotic medications.

BACKGROUND: Desired serotonin 5HT2 receptor pharmacology for treatment of psychoses is 5HT2A antagonism and/or 5HT2C agonism. No selective 5HT2A antagonist has been approved for psychosis and the only approved 5HT2C agonist (for obesity) also activates 5HT2A and 5HT2B receptors, which can lead to clinical complications. Studies herein tested the hypothesis that a dual-function 5HT2A antagonist/5HT2C agonist that does not activate 5HT2B receptors would be suitable for development as an antipsychotic drug, without liability for weight gain. METHODS: The novel compounds (+)- and (-)-trans-4-(4'-chlorophenyl)-N,N-dimethyl-2-aminotetralin (p-Cl-PAT) were synthesized, characterized in vitro for affinity and functional activity at human 5HT2 receptors, and administered by intraperitoneal (i.p.) and oral (gavage) routes to mice in behavioral paradigms that assessed antipsychotic efficacy and effects on feeding behavior. RESULTS: (+)- and (-)-p-Cl-PAT activated 5HT2C receptors, with (+)-p-Cl-PAT being 12-times more potent, consistent with its higher affinity across 5HT2 receptors. Neither p-Cl-PAT enantiomer activated 5HT2A or 5HT2B receptors at concentrations up to 300-times greater than their respective affinity (Ki), and (+)-p-Cl-PAT was shown to be a 5HT2A competitive antagonist. When administered i.p. or orally, (+)- and (-)-p-Cl-PAT attenuated the head-twitch response (HTR) in mice elicited by the 5HT2 agonist (-)-2,5-dimethoxy-4-iodoamphetamine (DOI) and reduced intake of a highly palatable food in non-food-deprived mice, with (+)-p-Cl-PAT being more potent across behavioral assays. CONCLUSIONS: The novel in vitro pharmacology of (+)-p-Cl-PAT (5HT2A antagonism/5HT2C agonism without activation of 5HT2B) translated in vivo to an orally-active drug candidate with preclinical efficacy to treat psychoses without liability for weight gain.

Action of a serotonergic anorectic in meal-fed mice working for food.

The aim of this study was to examine the effects of a serotonergic anorectic agent, dexnorfenfluramine (DNOR), on food intake in mice whose meals were constrained to specified periods each day and by effort. Mice were forced to adopt a human-like pattern of regular meals by making food available for four periods of 40 min/24-h period, mostly at night. They lived in behavior test chambers with a closed economy for food and were required to emit a fixed unit price (FUP) of either 2 or 25 nose pokes (FUP2, FUP25) to receive a 20 mg pellet of food. Once responding and intake were stable, mice were injected with a vehicle or DNOR (3 or 6 mg/kg) 1 h before a specified feeding opportunity. Food intake was dose-dependently suppressed at the next meal and to a greater extent when the cost of food was high (FUP25). Within a meal, the effect of the drug was the greatest in the first half of the available time. Therefore, the anorectic effect of DNOR was modified by the concurrent cost of food.

Effect of MTII on food intake and brain c-Fos in melanocortin-3, melanocortin-4, and double MC3 and MC4 receptor knockout mice.

Mice with genomic knockout of either melanocortin type 3 receptors (MC3R-/-), type 4 receptors (MC4R-/-) or knockout of both (double knockout, DKO) were tested for their anorectic response to the mixed MC3/4R agonist, MTII, injected into the anterior cerebral ventricle. Wild type (WT) mice showed a strong anorexia and, as expected, DKO were completely unresponsive to MTII. In contrast, both MC3R-/- and MC4R-/- showed a partial anorectic response. Induction of c-Fos immunoreactivity by MTII was examined in brain regions including paraventricular hypothalamus (PVN) and area postrema (AP). Compared with WT, MC4R-/- showed no activation in AP but showed normal activation in PVN, whereas MC3R-/- showed reduced activation in PVN but not in AP. RT-PCR analysis showed that hypothalamic mRNA for MC3R in MC4R-/- and for MC4R in MC3R-/- was unaltered from WT levels. These data suggest that both receptor subtypes are involved in the behavioral action of MTII, and that the critical receptors are in different brain regions.

Food demand and meal size in mice with single or combined disruption of melanocortin type 3 and 4 receptors.

Mice with homozygous genetic disruption of the melanocortin-4 receptor gene (MC4R-/-) are known to be hyperphagic and become obese, while those with disruption of the melanocortin-3 receptor gene (MC3R-/-) do not become markedly obese. The contribution of MC3R signaling in energy homeostasis remains little studied. In the present work, we compare MC3R-/- mice with wild-type (WT), MC4R-/-, and mice bearing disruption of both genes (double knockout, DKO) on select feeding and neuroanatomical dimensions. DKO mice were significantly more obese than MC4R-/-, whereas MC3R-/- weighed the same as WT. In a food demand protocol, DKO and MC4R-/- were hyperphagic at low unit costs for food, due primarily to increased meal size. However, at higher costs, their intake dropped below that of WT and MC3R-/-, indicating increased elasticity of food demand. To determine whether this higher elasticity was due to either the genotype or to the obese phenotype, the same food demand protocol was conducted in dietary obese C57BL6 mice. They showed similar elasticity to lean mice, suggesting that the effect is of genotypic origin. To assess whether the increased meal size in MC4R-/- and DKO might be due to reduced CCK signaling, we examined the acute anorectic effect of peripherally administered CCK and subsequently the induction of c-Fos immunoreactivity in select brain regions. The anorectic effect of CCK was comparable in MC4R-/-, DKO, and WT, but it was unexpectedly absent in MC3R-/-. CCK-induced c-Fos was lower in the paraventricular nucleus in MC3R-/- than the other genotypes. These data are discussed in terms of demand functions for food intake, MC receptors involved in feeding, and their relation to actions of gut hormones, such as CCK, and to obesity.

Effect of serotonergic anorectics on food intake and induction of Fos in brain of mice with disruption of melanocortin 3 and/or 4 receptors.

Previous studies have indicated that type 3 or 4 melanocortin receptors (MCR) are downstream of the critical anorectic action of drugs that stimulate 5-HT(2C) receptors. To characterize further the receptor types involved, we have studied the effect of serotonergic anorectics in mice with genomic disruption of either MC3R or MC4R, or their combined knockout. In a first experiment, we showed that wild type (WT) and MC4R-/- mice showed comparable inhibition of food intake following acute treatment with dexnorfenfluramine. In a second experiment using WAY-161503, a 5-HT receptor full agonist with selectivity for 2B and 2C subtypes, we found that MC4R-/- responded comparably to WT, while MC3R-/- had reduced sensitivity. Double receptor knockout (DKO) mice responded comparably to WT and MC4R-/-. Surprisingly, brain Fos-ir was not strongly induced in any brain region by WAY-16103 with the exception of the paraventricular nucleus of DKO. These data suggest that MC3Rs may be involved in the response to serotonergic anorectic agents, and more generally in control of food intake.

Effect of two types of environmental enrichment for singly housed mice on food intake and weight gain.

For animals that cannot be housed socially for scientific reasons, such as rodents on food intake-energy expenditure studies, the provision of environmental enrichment may alleviate stress. However, the influence of environmental enrichment on food intake and energy expenditure is unresolved. The authors tested the effects of two environmental enrichment options on food intake and weight gain in singly housed mice. Their results may be helpful in developing rodent enrichment programs.

Accurate caloric compensation in rats for electively consumed ethanol-beer or ethanol-polycose mixtures.

High elective intake of ethanol was achieved in rats by presenting ethanol in palatable vehicles. We simultaneously measured intake of food (chow) to assess the accuracy of caloric compensation for the energy in the alcoholic commodity. In the first study, we used beer; nonalcoholic beer was consumed in large amounts, and when 5% or 10% ethanol was added, intake amounted to approximately 10% of daily calories. In the second study, Polycose in either solution or a gel matrix was used as the palatable vehicle for ethanol. The intake of ethanol was even higher than in the beer study, particularly in the gel preparation. In all cases, both male and female rats showed accurate caloric compensation by a reduction in chow intake. In a final study, we showed that restricted time access to the Polycose-alcohol gel produced high elective intakes and substantial blood alcohol levels. Over 24 h, caloric compensation was again accurate. Thus, unlike some reports in humans, rats seem able to compensate accurately for alcohol calories and in particular when, as with most alcohol consumption by humans, these are presented in palatable vehicles.

LiCl-induced flavor avoidance compared between rats and mice using a nondeprivation protocol.

The present studies examine some parameters involved in flavor avoidance learning, using LiCl to induce malaise, in a novel nondeprivation protocol that allows direct comparison between rats and mice. The procedure involves daily presentation of a gelatin dessert that contains carbohydrate (Polycose) and a distinctive food flavor. Regular chow is additionally available at all times. Both rats and mice showed robust intakes of these gels with little change of gram intake as concentration of Polycose was varied in the range 2-30%; at the highest concentration, the caloric yield was approximately 7% of normal daily intake in both species. Rats that were injected on three occasions with LiCl (0.75 meq/kg) 1 h after consumption of a flavored gel formed a complete and sustained conditioned flavor avoidance (CFA). In a two-flavor discrimination protocol, in which a second flavor was followed by injections of saline, rats showed complete avoidance of the LiCl-paired flavor and partial avoidance of the saline-paired flavor. Mice injected on three occasions with LiCl (6 meq/kg) 1 h after intake of a flavored gel formed a partial CFA; a more complete CFA was formed when there was no delay between removal of the flavor and the injection. Using this no-delay protocol, mice, like rats, showed avoidance of a saline-paired flavor in a two-flavor discrimination protocol, and the CFA was strong when the dose of LiCl was reduced to that used in rats (0.75 meq/kg). In comparable protocols, mice thus are able to form complete CFAs using low doses of LiCl that are comparable to CFAs observed in rats, but the interval between flavor and sickness over which associative learning can occur may be shorter in mice.

Brain muscarinic receptor subtypes mediating water intake and Fos following cerebroventricular administration of bethanecol in rats.

RATIONALE: The brain regions and receptor subtypes involved in water intake following central cholinergic stimulation have been incompletely characterized. OBJECTIVES: To examine whether drinking and brain Fos-immunoreactivity (ir) induced in rats by central administration of bethanecol is reversed by either the preferential M1 antagonist pirenzepine, the M3 antagonist 4-DAMP, or their combination. METHODS: Male Sprague-Dawley rats were surgically implanted with cerebroventricular cannulae. The muscarinic agonist, bethanecol was used as the dipsogenic agent. Either nonselective (atropine) or selective muscarinic receptor antagonists were injected together with bethanecol to determine blockade of drinking. In parallel studies, Fos-ir was assessed in discrete brain regions. RESULTS: Bethanecol-induced drinking was completely blocked by atropine or by a combination of pirenzepine and 4-DAMP; these latter antagonists alone produced sub-total inhibition of drinking. In contrast, water intake induced by angiotensin II was unaffected by combination of pirenzepine and 4-DAMP. Fos-ir was induced by bethanecol in many brain regions previously implicated in body fluid regulation, including subfornical organ and the magnocellular supraoptic and paraventricular hypothalamic nuclei. Induced Fos-ir was substantially but not completely prevented by co-injection of either pirenzepine or 4-DAMP, but their combination did not seem markedly more effective than either alone. CONCLUSIONS: Drinking induced by brain muscarinic receptor stimulation seems to proceed by a combination of M1 and M3 receptor subtypes. Drinking induced by angiotensin II occurs independently of this mechanism. Fos-ir induced in fluid-related brain regions by bethanecol either uses additional receptor type(s) or is less easily blocked than drinking behavior.